This is Project 3 of the consortium proposal focused on the manipulation of hematopoietic and mesenchymal stem cells for the control of donor-specific alloreactivity in Cynomolgus monkeys. This project will define the role of regulatory T cells and cytokines in allograft acceptance of allogeneic islet and kidney transplantation. A central role of T immune regulatory cells in the maintenance of transplant tolerance is an emerging concept requiring critical evaluation in non-human primate models of allograft transplantation. Early studies with murine transplantation models had suggested that suppression of Thl cytokine responses, primarily IFN-gamma, and enhancement of Th2 responses, primarily IL-4, were critical for tolerance. These observations were consistent with the prevailing concepts that IL-4 could negatively regulate Thl cytokine responses and that IFN-gamma was proinflammatory. More recently the cytokines IL-l0 and TGF-beta, which have anti-inflammatory properties, have been implicated in the maintenance of tolerance. Specifically, a subset of CD4+ T cells, designated T regulatory cells, has been demonstrated to inhibit antigen-specific immune responses through the secretion of IL-l0 and TGF-beta and expression of CTLA-4. T regulatory cells have been identified in models of autoimmunity and allograft tolerance in mice and humans. This has led to a compelling new hypothesis of allograft tolerance in which T regulatory cells, producing IL-l0 and/or TGFbeta, are the primary mediators of peripheral allograft tolerance. Under this hypothesis, the tolerant state is a dynamic one in which proinflammatory cells are held in check by T regulatory cells. Our murine model of tolerance involving co transplantation of intact active bone fragments (JAB) and transient costimulation blockade results in stable, long-term tolerance. Investigations into the mechanism maintaining the tolerant state suggest a role for IL-l0-producing T regulatory cells. Preliminary results in a Cynomolgus monkey that accepted an allogeneic kidney graft for more than 145 days are also consistent with a hypothesis that, in immunologically intact recipients, a dominance of T regulatory cells over proinflammatory T cells produces long-term allograft acceptance. Here we propose to test whether allograft tolerance induced by non-myeloablative regimens in the setting of allogeneic islet and renal transplantation in non-human primates depends on a dominant T regulatory cell population. We also propose to test whether bone marrow- derived mesenchymal stem cells can enhance the development of T regulatory cells. We anticipate that these mechanistic studies will lead to an increased understanding of the roles of cytokines and T regulatory cells in allograft acceptance and tolerance. Our proposed studies will also evaluate ex vivo cytokine assays as biomarkers of immune tolerance.